Glutathione modulates the level of free radicals produced in UVA-irradiated cells

We have developed an assay to detect reactive oxygen species (ROS) generate d by UVA radiation utilising chemical probes which become fluorescent upon oxidation. Using a human bladder carcinoma cell line (MGH-UI) and spontaneo usly immortalised keratinocytes (HaCaT), we have shown a UVA (narrow band 3 65+/-5 Mn) dose-dependent increase in fluorescence by flow cytometry follow ing loading of the cells with either dihydrorhodamine 123 (DHR) or 2',7'-di chlorodihydrofluorescein diacetate (DCFH-DA). The UVA response of both DHR and DCFH was enhanced by elevation of intracellular levels of the photosens itiser protoporphyrin IX by incubation for 2.5 h with 5-aminolaevulinic aci d. Depletion of the antioxidant glutathione (GSH) using the inhibitor D,L-b uthionine-sulphoximine (BSO), resulted in an increase in the UVA induced fl uorescence of DCF but not of rhodamine 123. Conversely, raising intracellul ar GSH levels with N-acetyl cysteine (NAC) had relatively little protective effect in terms of degree of induced fluorescence. (C) 2000 Elsevier Scien ce S.A. All rights reserved.